Activation of the leu-500 Promoter by a Reversed Polarity tetA Gene

The leu-500 promoter is inactivated by a mutation in the −10 region but can be activated in topA Escherichia coli and Salmonella strains. We have found that the tetA gene plays a vital role in thetopA-dependent activation of a plasmid-borneleu-500 promoter. In previous studies, theleu-500 promoter and tetA gene have been arranged divergently. In this study we have reversed the polarity of the tetA gene, thus locating the leu-500promoter at the 3′ end of tetA. Despite being formally located in the downstream region of tetA, theleu-500 promoter is equally well activated in atopA strain in this environment, even though it is 1.6 kilobase pairs away from the promoter of the reversed tetAgene. Activation of the leu-500 promoter depends on transcription and translation of tetA but is largely insensitive to the function of other transcription units on the plasmid. These results require a change in viewpoint of the role oftetA, from local to global supercoiling. We conclude that transcription of the tetA gene is the main generator of transcription-induced supercoiling that activates theleu-500 promoter. Unbalanced relaxation of this supercoiling leads to a net increase in the negative linking difference of the plasmid globally, and there is a linear correlation between the change in global plasmid topology and the activation of theleu-500 promoter. Thus the leu-500 promoter appears to respond to the negative supercoiling of the plasmid overall.